Adult bone marrow (BM) is the most common source of mesenchymal stem/progenitor cells (MSCs), (also called Mesenchymal Stromal Cells1) which are functionally defined by their capability of differentiating into the skeletal tissues: bone2-4, cartilage5-7, fat8 and muscle9 in vitro. MSCs are classically distinguished from the heterogeneous milieu of cells through adhesion to tissue culture plastic and the formation of colony unit-fibroblasts (CFU-Fs), the frequency of which are 1:100,000-1:500,000 nucleated cells in adult marrow10, and studies have now identified a suite of markers with which MSCs are categorized10,11. This low proportion of MSCs leads to the necessity of culture expansion and selection before use to attain the appropriate cell numbers for any kind of cellular therapy. There are other emerging sources of MSCs such as: adipose tissue12, trabecular bone13 and fetal liver14 which have a CFU-F frequency of: 1:3215, 1:63613 and 1:88,49514 respectively. While adipose tissue does appear to have the highest frequency of progenitors, the doubling time of those cells ranges between 3.6 to 4.4 days15, and the extraction procedure is complicated, invasive, and lengthy12. Harvesting trabecular bone results in low cell yield (89×106 cells/gram of bone from young donors13), especially when combined with the CFU-F frequency; and is extremely invasive resulting in donor site morbidity.
Unique among these new sources of MSCs are human umbilical cord perivascular cells (HUCPVCs), which are an easily accessible, highly proliferative source of cells with a population doubling time of 20 hours (dependent on serum)16. The frequency of CFU-Fs in HUCPVCs is 1:300 at passage 0 but increases to 1:3 at passage 117, which is orders of magnitude higher than bone marrow16. Therefore HUCPVCs represent a population of cells with an extremely high proportion of MSCs which proceed to divide very quickly, thus making them an excellent candidate for clinical mesenchymal therapies. These cells have been used in various assays to determine their marker expression phenotype and differentiation potential16,18, and have been found to be either bioequivalent to, or perform better than, BM-MSCs.
In addition to their ability to differentiate, MSCs also have potential immunological uses as BM-MSCs have been shown to be both immunoprivileged and immunomodulatory19-21. These terms refer to a cell's ability to evade recognition from a mismatched host's immune system, and the ability to mitigate an ongoing response by that system, respectively. MSCs from several sources other than bone marrow have been tested for their immunogenicity in in vitro cultures. MSCs from adipose tissue derived from adult dermolipectomies were shown to be capable of both immunoprivilege and immunomodulation in vitro22, whereas fetal liver cells were found to be capable of avoiding a mismatched immune response, however they were not able to modulate alloreactivity caused by two mismatched populations of lymphocytes23,24. Thus, the source of MSCs directly affects those cells' immunogenic capabilities.
This in vitro work has begun to be validated in the clinical setting; for example, a boy was rescued from severe acute graft vs. host disease (GvHD) by transfusion of haploidentical bone marrow MSCs from his mother25. One year post treatment, in comparison to a cohort of patients suffering from the same level of severity of the disease, he was the only one alive. Since this initial patient, a suite of 8 patients have been treated with BM-MSCs, of which 6 showed a complete remission of symptoms26. Allogeneic BM-MSCs have also been used in Crohn's Disease to treat patients who are refractory to current treatments, and this treatment is currently in clinical trials in the United States27,28. Fetal liver MSCs have shown efficacy in the early treatment of osteogenesis imperfecta (OI). MSCs from a male fetal liver were transplanted into an unrelated 32 week female fetus with severe OI, who had suffered several intrauterine fractures29. Following the transplantation, the remainder of the pregnancy proceeded normally, and there were no further fractures. This patient has been followed up to 2 years after birth, and the child has shown a normal growth curve and has suffered only 3 fractures. Using an XY-specific probe, the patient was found to have 0.3% engraftment in a bone biopsy.
In addition to undifferentiated cells, osteogenically induced rabbit BM-MSCs were found to be immunoprivileged and immunomodulatory in vitro, but when transplanted in vivo the immunomodulatory capacity was lost30. This would not affect the function of the cells however; as they only require protection from an immune response in order to fulfill their role. In a more involved induction, murine bone marrow MSCs were manipulated to release erythropoietin and implanted in mice, which resulted in significantly less engraftment compared to un-manipulated controls31. Thus, manipulation of MSCs can lead to their loss of immunomodulation and/or immunoprivilege and can be crucial to the survival and function of the graft.
There is evidence to support that the immunoprivilege of MSCs transcends species barriers, and they can be used xenogeneically. This was first demonstrated by Bartholomew et al who used human BM-MSCs in baboons, and showed enhanced skin graft survival21. While the end result of this study was positive, the specific fate of the administered cells was not determined. Wang et al. have utilized GFP transfected cells and histological analyses to studied the survival of xenogeneic BM-MSCs, and showed that the cells survive up to the 11 week timepoint without immunosuppression, however there was an increased host immune reaction32. MSCs have also been reported to survive in xenogeneic transplantations in two cardiac models33,34. In preliminary work with HUCPVCs, the cells were delivered peritoneally in permeable chambers. After 3 weeks, there was no noticeable inflammation noted upon macroscopic visualization35. This is encouraging preliminary work indicating the potential for not only the immunoprivilege of HUCPVCs, but also for their ability to test them in animal models without rejection.
The inventors investigated the immunoprivileged and immunomodulatory properties of HUCPVCs in vitro by conducting both: co-cultures of HUCPVCs with unmatched lymphocytes, and mixed lymphocyte cultures (MLCs) populated by two HLA mismatched donors. Also studied were HUCPVC death, lymphocyte proliferation and activation with varying levels of HUCPVCs present in both naïve and activated lymphocyte environments. In addition, the necessity for cell contact for the observation of immunological effects was investigated.